The goalof the proposed research is to dissect the molecular and genetic pathway involving Pax6 and Eya in eye development and to determine the functional roles of Eya proteins. The first specific aim focuses on the regulation of Eya by Pax6, specifically whether Pax6 is a direct or indirect regulation of Eya expression. Expression of reporter constructs in vitro and in vivo will be used to determine Eya promoter regions regulated by Pax6. A binding site selection assay will be use to screen for Pax6 binding regions within Eya promoter fragments shown to drive expression in cultured cells. Candidate regions will be molecularly characterized. Candidate Pax6 binding regions will be cloned ahead of reporter constructs and used to make transgenic animals to test the ability of the target sites to recapitulate the expression pattern of the endogenous Eya gene. The evolutionary conservation of Pax6/Eya regulation will be assessed and making transgenic files in which candidate Eya regulatory regions are place upstream of a lacZ reporter and examining staining patterns in developing eye discs. Pax6-dependence of the transgenes will be observed in hypomorphic alleles of the Drosophila Pax6 homolog, eyeless.